PROJECT SUMMARY/ABSTRACT Mitochondrial gene expression is an oft-cited target for antiparasitic drugs to combat the dangerous, insect- transmitted trypanosome parasites. Mitochondrial gene expression regulation, though likely crucial to parasite survival, is not well understood. The various steps in mitochondrial gene expression have been elucidated. However we still do not know the control mechanisms used to generate the proper ratios of mature mitochondrial mRNAs appropriate for each parasite life stage. The proposed project focuses on a piece of this puzzle: the populations of mitochondrial RNA (mtRNA) 3' non-encoded adenine- and uridine-containing nucleotide ?tails? that are thought to regulate the post-transcriptional events that determine trypanosome mtRNA fates. The proposal considers that the function of tails may not be universal as in current models but rather that they play specific roles depending on their characteristics. Newly obtained data demonstrate a previously unrecognized range of compositional and length variations that differ between tail populations of the 18 encoded mtRNAs and also between tail populations in the insect and mammalian parasite life stages. However, the control mechanisms by which tail differences are established, are, with one major exception, entirely unknown, and the focus of this study. This proposal's specific hypothesis is that differential actions of two non-canonical poly(A) polymerases are responsible for transcript- and life stage-specific differences in trypanosome mtRNA tail compositions. The Aim 1 focus is the observed transcript-to-transcript variations in tail population characteristics, and Aim 1 hypothesis is that transcript-specific targeting of a minor polymerase (KPAP2) is responsible for these variations. The role of KPAP2 in achieving transcript-specific differences in tail compositions will be determined. Aim 2 will examine how stage-specific differences in tail populations are achieved. The primary polymerase KPAP1 can carry methylmarks on certain arginine residues that are likely to impact its RNA or protein interactions, thereby impacting function. The degree to which KPAP1 arginine methylation impacts tail addition in each life stage will be determined; the hypothesis is that life-stage specific differential methylation is the cause of at least some life stage differences in tail characteristics. These discoveries will be significant because they will provide us a means to perturb the differences in tail compositions between and within life stages and observe the impact various other post-transcriptional processes of trypanosome mitochondrial gene expression. In addition to the perspective of tail variation as a gene expression regulator, the other innovation of this proposal is technical: it utilizes a new technique to collect large populations of tail sequences. In summary, this work will uncover control mechanisms that result in differences in mtRNA tails that may ultimately be responsible for differences in relative abundances of mtRNAs within a single life stage and between life stages.